Peptide-Mediated Non-Covalent Delivery of Active Agents Across the Blood-Brain Barrier

ABSTRACT

The peptides described herein can function as carrier peptides. These peptides can associate with (e.g., non-covalently bind) biologically active molecules or imaging agents to transport the biologically active molecules or imaging across the blood-brain barrier. In some cases, such transport may increase the effectiveness of the biological molecules or imaging agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/422,827, filed Feb. 2, 2017, which is a continuation of U.S.application Ser. No. 14/281,389, filed May 19, 2014 (now U.S. Pat. No.9,597,408), which is a divisional of U.S. application Ser. No.13/383,710, filed Jan. 12, 2012 (Abandoned), which is a National Stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2010/041924, filed Jul. 14, 2010, which claims priority to U.S.Provisional Application Ser. No. 61/225,412, filed on Jul. 14, 2009,which are incorporated by reference in their entirety herein.

TECHNICAL FIELD

This disclosure relates to carrier peptides, including compositions andmethods of using the same. In particular, the disclosure relates tocarrier peptides capable of delivering active agents across theblood-brain barrier and compositions and methods of using the same.

BACKGROUND

The blood-brain barrier (BBB) prevents most macromolecules (e.g., DNA,RNA, and polypeptides) and many small molecules from entering the brain.The BBB is principally composed of specialized endothelial cells withhighly restrictive tight junctions, consequently, passage of substances,small and large, from the blood into the central nervous system iscontrolled by the BBB. This structure makes treatment and management ofpatients with neurological diseases and disorders (e.g., brain cancerand Alzheimer's disease) difficult as many therapeutic agents cannot bedelivered across the BBB with desirable efficiency.

SUMMARY

The peptides described herein can function as carrier peptides. Thesepeptides can associate with (e.g., non-covalently bind) biologicallyactive molecules and imaging agents to transport the biologically activemolecules and imaging agents across the blood-brain barrier. In somecases, such transport may increase the effectiveness of the biologicalmolecules and imaging agents.

A carrier peptide, as described herein, can include the sequence:

X_(n)—[B]_(m)

or a pharmaceutically acceptable salt thereof, wherein:X is a hydrophilic amino acid;B is a blood-brain barrier agent;n is an integer from 4 to 50; andm is integer from 1 to 3.

In some embodiments, the blood-brain barrier agent is notL-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R (SEQ ID NO:141).

A hydrophilic amino acid (X) can be chosen from arginine, asparagine,aspartic acid, glutamic acid, glutamine, histidine, lysine, serine,threonine, tyrosine, or a combination thereof. For example, in someembodiments, X_(n) can comprise ten lysines and six arginines; sixlysines and sixteen arginines; eight lysines, eight arginines, and eighthistidines. In some embodiments, X is lysine.

The variable n is an integer ranging from 4 to 50 (e.g., 4, 6, 8, 10,12, 16, 20, 24, 26, 28, 32, 36, 40, 42, 44, 48, and 50). In someembodiments, n is chosen from 4, 8, 12, 16, and 20. In some embodiments,n is 16. In some embodiments, m is 1.

The blood-brain barrier agent can be a receptor binding domain of anapolipoprotein. For example, the receptor binding domain of anapolipoprotein can be chosen from the receptor binding domain of ApoA,ApoB, ApoC, ApoD, ApoE, ApoE2, ApoE3, and ApoE4. In some embodiments,the receptor binding domain of an apolipoprotein is chosen from thereceptor binding domain of ApoB and ApoE.

The blood-brain barrier agent can also include a polypeptide sequencehaving at least 80% sequence identity to:

(SEQ ID NO: 13) L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 14)S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 15)Y-P-A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R-H-Y-L-N-L-V-T-R-Q-R-Y*; (SEQ ID NO: 16)A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R-H-Y- L-N-L-V-T-R-Q-R-Y*;(SEQ ID NO: 17) Y-P-S-D-P-D-N-P-G-E-D-A-P-A-E-D-L-A-R-Y-Y-S-A-L-R-H-Y-I-N-L-I-T-R-Q-R-Y*; or (SEQ ID NO: 18)A-P-L-E-P-V-Y-P-G-D-D-A-T-P-E-Q-M-A-Q-Y-A-A-E-L-R-R-Y-I-N-M-L-T-R-P-R-Y*,wherein Y* is tyrosine or a tyrosine derivative. In some embodiments,the polypeptide is less than 100 amino acids in length.

Examples of a carrier peptide can include:

(SEQ ID NO: 19) K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A;(SEQ ID NO: 20) K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 21)K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R- K-R-L-L-R-D-A;(SEQ ID NO: 22) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 24)K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 25)K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 26)K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G- S-H; (SEQ ID NO: 27)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K- F-V-E-G-S-H; and(SEQ ID NO: 28) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S- L-S-N-K-F-V-E-G-S-H.

Further provided herein is a complex, or a pharmaceutically acceptablesalt thereof, having a biologically active molecule or an imaging agentassociated with a carrier peptide, as described above. In someembodiments, the biologically active molecule or imaging agent isnon-covalently bound to the carrier peptide.

An imaging agent can be any chemical or substance which is used toprovide the signal or contrast in imaging. For example, the imagingagent can be ¹²⁵I-IgG or magnevist. A biologically active molecule caninclude a polypeptide; oligonucleotide; plasmid; small molecule;antibody; antibody fragment; carbohydrate; polysaccharide; lipid;glycolipid; antigen; and antigenic peptide. In some embodiments, thebiologically active molecule is chosen from a: polypeptide;oligonucleotide; and plasmid. An oligonucleotide can include a codingDNA sequence; antisense DNA sequence; mRNA, antisense RNA sequence;RNAi; and siRNA. In some embodiments, the biologically active moleculeis a small molecule, for example, a therapeutic agent.

The carrier peptides and complexes of carrier peptides and biologicallyactive agents or imaging agents have various uses and can be used invarious methods. For example, provided herein is a method oftransporting a biologically active molecule or imaging agent across theblood-brain barrier of a subject. The method can include administeringto the subject a complex, or a pharmaceutically acceptable salt thereof,having the biologically active molecule or imaging agent associated witha carrier peptide.

Also provided is a method of treating a brain disorder in a subject. Themethod can include administering to the subject a complex, or apharmaceutically acceptable salt thereof, having a biologically activeagent associated with a carrier peptide. The brain disorder can bechosen from meningitis, epilepsy, multiple sclerosis, neuromyelitisoptica, late-stage neurological trypanosomiasis, Parkinson's,progressive multifocal leukoencephalopathy, De Vivo disease, Alzheimer'sdisease, HIV Encephalitis, and cancer.

This disclosure further provides a method of imaging the central nervoussystem of a subject. The method can include administering to the subjecta complex, or a pharmaceutically acceptable salt thereof, comprising animaging agent associated with a carrier peptide; and imaging the centralnervous system of the subject.

Pharmaceutical compositions are also disclosed. A pharmaceuticalcomposition can include a complex, or a pharmaceutically acceptable saltthereof, having a biologically active molecule or imaging agent, acarrier peptide, and a pharmaceutically acceptable carrier.

Finally, kits are also provided. A kit can include a carrier peptide. Insome embodiments, the kit further includes a biologically activemolecule or an imaging agent.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a series of images illustrating the differences betweendelivery of Alexa-IgG complexed to K₁₆ApoE (SEQ ID NO: 22) into brainand uncomplexed Alexa-IgG.

FIG. 2 illustrates the accumulation of radioactive IgG 4.1 in mousebrain aided by K₁₆ApoE (SEQ ID NO: 22).

FIG. 3 shows delivery of K16APoE-mediated (SEQ ID NO: 22)beta-galactosidase in brain (Top) and in other organs (Bottom). In oneexperiment, beta-galactosidase was injected in mice mixed with (FIG.3A′) and without K16ApoE (SEQ ID NO: 22) (FIG. 3A), brain slices madesix hours after injection were stained for enzyme activity (top). In aseparate related experiment, slides were made from different organs andstained for beta-galactosidase activity after delivery without (FIG.3B-G) and with K16ApoE (SEQ ID NO: 22) (FIG. 3B′-G′), B—brain; C—heart,D—kidney, E—lung, F—liver, G—spleen.

FIG. 4 shows the evaluation of K16ApoE-mediated delivery of IgG (A) andIgM (B) in mice brains by microSPECT. Imaging was done at 1 hourintervals up to six hours, after which cardiac perfusion was performed,and final imaging carried out 30 minutes after perfusion. Left bars—withK16ApoE (SEQ ID NO: 22); Right bars—no K16ApoE (SEQ ID NO: 22).

FIG. 5 illustrates labeling of amyloid plaques with a plaque-specificantibody delivered via K16ApoE (SEQ ID NO: 22) in brains of mice modelsof Alzheimer's disease. Two mice with Alzheimer's disease were used: onerepresented by A, C and E, while the other is represented by B, D and F.A, C and E represent adjacent brain sections from one mouse, whereas B,D and F represent adjacent brain sections from another mouse. A,B—thioflavine S staining; C, D—immunostaining to identify plaques usingthe 4G8 as the primary antibody, and an anti-mouse antibody as thesecondary antibody; E, F—immunostaining using the secondary antibodyonly. The 4G8 IgG was injected in the first mouse (left panel) withoutK16ApoE (SEQ ID NO: 22), while the second mouse (right panel) receivedinjection of the IgG mixed with K16ApoE (SEQ ID NO: 22).

FIG. 6 shows the evaluation of the integrity of the BBB after injectionof K16ApoE (SEQ ID NO: 22). Each mouse (represented by A through G)received an injection of K16ApoE (SEQ ID NO: 22), followed by injectionof β-galactosidase at the indicated times. Brain slices were made 4hours after β-galactosidase injection and were followed by staining foractivity of the enzyme. A—β-galactosidase was injected 1 minute afterK16ApoE (SEQ ID NO: 22) injection. B—β-galactosidase was injected 5minutes after K16ApoE (SEQ ID NO: 22) injection. C—β-galactosidase wasinjected 10 minutes after K16ApoE (SEQ ID NO: 22) injection.D—β-galactosidase was injected 30 minutes after K16ApoE (SEQ ID NO: 22)injection. E—β-galactosidase was injected 1 hour after K16ApoE (SEQ IDNO: 22) injection. F—β-galactosidase was injected 2 hours after K16ApoE(SEQ ID NO: 22) injection. G—β-galactosidase was injected 4 hours afterK16ApoE (SEQ ID NO: 22) injection. H—positive control (β-galactosidasemixed with K16ApoE (SEQ ID NO: 22) was injected, brain slices were made4 hours after the injection and proceeded for staining for enzymeactivity).

FIG. 7 demonstrates the need for chemical linking of the K16 (SEQ ID NO:4) and the ApoE peptide moieties for non-covalent delivery across theBBB. Delivery of β-galactosidase in brain with K16 peptide (SEQ ID NO:4) alone (A), with ApoE peptide only (B), with K16 peptide (SEQ ID NO:4)+ApoE peptide (C), and with K16APoE peptide (SEQ ID NO: 22) (D).

FIG. 8 illustrates the testing mutant ApoE peptides for their potentialas non-covalent transporters of proteins across the BBB. A—no peptide,no β-gal; B—K16ApoE (SEQ ID NO: 22)+β-gal; C—K16L3N7 (SEQ ID NO:126)+β-gal; D—K16L3L20 (SEQ ID NO: 127)+β-gal; E—K16N7L20 (SEQ ID NO:128)+β-gal.

FIG. 9 shows a time course study of β-galactosidase delivery in mousebrain with K16ApoE (SEQ ID NO: 22). A—β-galactosidase, no peptide, 6 h;B—β-galactosidase+ peptide, 1 h; C—β-galactosidase+ peptide, 2 h;D—β-galactosidase+ peptide, 5 h; E—β-galactosidase+ peptide, 10 h.

DETAILED DESCRIPTION

The peptides described herein can function as carrier peptides. Thesepeptides can associate with (e.g., non-covalently bind) biologicallyactive molecules and imaging agents to transport the biologically activemolecules and imaging agents across the blood-brain barrier. In somecases, such transport may increase the effectiveness of the biologicalmolecules and imaging agents.

Carrier Peptides

Provided herein are carrier peptides having the following sequence:

X_(n)—[B]_(m),

or a pharmaceutically acceptable salt, thereof, wherein:X is a hydrophilic amino acid;B is a blood-brain barrier agent;n is an integer from 4 to 50; andm is integer from 1 to 3.

In some embodiments, the blood-brain barrier is notL-R-K-L-R-K-R-L-L-R-L-R-K-L-R-K-R-L-L-R (SEQ ID NO: 141).

A hydrophilic amino acid can be chosen from: arginine, asparagine,aspartic acid, glutamic acid, glutamine, histidine, lysine, serine,threonine, tyrosine, and combinations and non-natural derivativesthereof. In some embodiments, a hydrophilic amino acid can be chosenfrom lysine or a non-natural lysine derivative, arginine or anon-natural arginine derivative, and combinations thereof. In someembodiments, the hydrophilic amino acid is lysine. Non-limiting examplesof X_(n) can include KKKK (SEQ ID NO:1); KKKKKKKK (SEQ ID NO:2);KKKKKKKKKKKK (SEQ ID NO:3); KKKKKKKKKKKKKKKK (SEQ ID NO:4); RRRR (SEQ IDNO:5); RRRRRRRR (SEQ ID NO:6); RRRRRRRRRRRR (SEQ ID NO:7);RRRRRRRRRRRRRRRR (SEQ ID NO:8); KRKR (SEQ ID NO:9); KKKR (SEQ ID NO:10);KKKRRRKKKRRR (SEQ ID NO:11); and KKKKRRRRKKKKRRRR (SEQ ID NO:12).

The variable n is an integer ranging from 4 to 50 (e.g., 4, 6, 8, 10,12, 16, 20, 24, 26, 28, 32, 36, 40, 42, 44, 48, and 50). For example, ncan range from 4 to 20. In some embodiments, n is chosen from 4, 8, 12,16, and 20. For example, n can be 16. In some embodiments, m is 1.

A blood-brain barrier agent, as used herein, is any polypeptide ornon-polypeptide ligand that can cross the blood-brain barrier. In someembodiments, a blood-brain barrier agent has a cognate receptor on braincells or can bind to such receptors. In some embodiments, theblood-brain barrier agent comprises a transferring-receptor binding siteof a transferrin. In some embodiments, the blood-brain barrier agentcomprises a receptor binding domain of an apolipoprotein. A receptorbinding domain of an apolipoprotein, for example, can be chosen from thereceptor binding domain of ApoA, ApoB, ApoC, ApoD, ApoE, ApoE2, ApoE3,ApoE4, and combinations thereof. In some embodiments, the receptorbinding domain of an apoliprotein is chosen from the receptor bindingdomain of ApoB and ApoE.

In some embodiments, the blood-brain barrier agent comprises a sequencehaving at least 80% (e.g., at least 85%; at least 90%; at least 92%; atleast 95%; at least 98%; and at least 99%) sequence identity to:

(SEQ ID NO: 13) L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 14)S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 15)Y-P-A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R-H-Y-L-N-L-V-T-R-Q-R-Y*; (SEQ ID NO: 16)A-K-P-E-A-P-G-E-D-A-S-P-E-E-L-S-R-Y-Y-A-S-L-R-H-Y- L-N-L-V-T-R-Q-R-Y*;(SEQ ID NO: 17) Y-P-S-D-P-D-N-P-G-E-D-A-P-A-E-D-L-A-R-Y-Y-S-A-L-R-H-Y-I-N-L-I-T-R-Q-R-Y*; or (SEQ ID NO: 18)A-P-L-E-P-V-Y-P-G-D-D-A-T-P-E-Q-M-A-Q-Y-A-A-E-L-R-R-Y-I-N-M-L-T-R-P-R-Y*,wherein Y* is tyrosine or a tyrosine derivative (e.g., an amidatedtyrosine). See, e.g., Ballantyne, G. H., Obesity Surgery, 16:651-6582006.

In some embodiments, the blood-brain barrier agent includes apolypeptide comprising the following sequence:

(SEQ ID NO: 29) L-R-X1-R-X2-X3-X4-H-L-R-X5-X6-X7-K-R-L-X8-R-D-X9wherein:X1 is selected from the group consisting of A, L, S, and V;X2 is selected from the group consisting of L and M;X3 is selected from the group consisting of A and S;X4 is selected from the group consisting of N, S, and T;X5 is selected from the group consisting of K and N;X6 is selected from the group consisting of L, M, and V;X7 is selected from the group consisting of R and P;X8 is selected from the group consisting of L and M; andX9 is selected from the group consisting of A and L.Non-limiting examples of a blood-brain barrier agents according to thissequence include:

(SEQ ID NO: 13) L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 30)L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 31)L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 32)L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 33)L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 34)L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 35)L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 36)L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 37)L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 38)L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 39)L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 40)L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 41)L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 42)L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 43)L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 44)L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 45)L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 129)L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; and (SEQ ID NO: 130)L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L.

In some embodiments, the blood-brain barrier agent is less than 100amino acids in length (e.g., less than 90 amino acids in length; lessthan 80 amino acids in length; less than 75 amino acids in length; lessthan 70 amino acids in length; less than 65 amino acids in length; lessthan 62 amino acids in length; less than 60 amino acids in length; lessthan 55 amino acids in length; less than 50 amino acids in length; andless than 45 amino acids in length).

“Percent sequence identity” refers to the degree of sequence identitybetween any given reference sequence, e.g., SEQ ID NO:13, and acandidate blood-brain barrier agent sequence. A candidate sequencetypically has a length that is from 80 percent to 200 percent of thelength of the reference sequence (e.g., 82, 85, 87, 89, 90, 93, 95, 97,99, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, or 200percent of the length of the reference sequence). A percent identity forany candidate nucleic acid or polypeptide relative to a referencenucleic acid or polypeptide can be determined as follows. A referencesequence (e.g., a nucleic acid sequence or an amino acid sequence) isaligned to one or more candidate sequences using the computer programClustalW (version 1.83, default parameters), which allows alignments ofnucleic acid or polypeptide sequences to be carried out across theirentire length (global alignment). Chenna et al., Nucleic Acids Res.,31(13):3497-500 (2003).

ClustalW calculates the best match between a reference and one or morecandidate sequences, and aligns them so that identities, similaritiesand differences can be determined. Gaps of one or more residues can beinserted into a reference sequence, a candidate sequence, or both, tomaximize sequence alignments. For fast pairwise alignment of nucleicacid sequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10. gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of peptide sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of peptidesequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10. gap extension penalty: 0.5; hydrophilic gaps: on;hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys;residue-specific gap penalties: on. The ClustalW output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launchersite (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and atthe European Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

To determine percent identity of a candidate nucleic acid or amino acidsequence to a reference sequence, the sequences are aligned usingClustalW, the number of identical matches in the alignment is divided bythe length of the reference sequence, and the result is multiplied by100. It is noted that the percent identity value can be rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded upto 78.2.

The variable m is an integer from 1 to 3. In some embodiments, m is 1.

In some embodiments, a carrier peptide can be chosen from:

(SEQ ID NO: 19) K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A;(SEQ ID NO: 20) K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 21)K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R- K-R-L-L-R-D-A;(SEQ ID NO: 22) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 24)K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 25)K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G-S-H; (SEQ ID NO: 26)K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K-F-V-E-G- S-H; (SEQ ID NO: 27)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S-L-S-N-K- F-V-E-G-S-H; and(SEQ ID NO: 28) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-S-S-V-I-D-A-L-Q-Y-K-L-E-G-T-T-R-L-T-R-K-R-G-L-K-L-A-T-A-L-S- L-S-N-K-F-V-E-G-S-H.

In some embodiments, a carrier peptide can be chosen from:

(SEQ ID NO: 142) [X]_(n)-L-R-X1-R-X2-X3-X4-H-L-R-X5-X6-X7-K-R-L-X8-R-D-X9wherein:X is a hydrophilic amino acid;n is an integer from 4 to 20;X1 is selected from the group consisting of A, L, S, and V;X2 is selected from the group consisting of L and M;X3 is selected from the group consisting of A and S;X4 is selected from the group consisting of N, S, and T;X5 is selected from the group consisting of K and N;X6 is selected from the group consisting of L, M, and V;X7 is selected from the group consisting of R and P;X8 is selected from the group consisting of L and M; andX9 is selected from the group consisting of A and L.Non-limiting examples of a blood-brain barrier agents according to thissequence include:

(SEQ ID NO: 19) K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A;(SEQ ID NO: 20) K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 21)K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R- K-R-L-L-R-D-A;(SEQ ID NO: 22) K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 46)K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 47)K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 48) K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 49)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 50)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A ; (SEQ ID NO: 51)K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 52)K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 53) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 54)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 55)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 56)K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 57)K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 58) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 59)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 60)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 61)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 62)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L- R-D-A;(SEQ ID NO: 63) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 64)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 65)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 66)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 67)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M- R-D-A;(SEQ ID NO: 68) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 69)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 70)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 71)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 72)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 73) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 74)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 75)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 76)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 77)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L- R-D-A;(SEQ ID NO: 78) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 79)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 80)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 81)K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 82)K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 83) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 84)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 85)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 86)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 87)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L- R-D-A;(SEQ ID NO: 88) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 89)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 90)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 91)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 92)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L- R-D-A;(SEQ ID NO: 93) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 94)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 95)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 96)K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 97)K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 98) K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 99)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 100)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 101)K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 102)K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M- R-D-A;(SEQ ID NO: 103) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 104)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 105)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 106)K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 107)K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M- R-D-A;(SEQ ID NO: 108) K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 109)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 110)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 111)K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 112)K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 113) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 114)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 115)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 116)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 117)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M- R-D-A;(SEQ ID NO: 118) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 119)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 120)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 121)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 122)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L- R-D-A;(SEQ ID NO: 123) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 124)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 125)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 131)K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 132)K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L- R-D-L;(SEQ ID NO: 133) K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 134)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 135)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 136)K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 137)K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L- R-D-L;(SEQ ID NO: 138) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 139)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; or (SEQ ID NO: 140)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L.

Complexes

Also provided herein are complexes having a biologically active moleculeor an imaging agent associated with a carrier peptide as describedherein. In some embodiments, the biologically active molecule or imagingagent is non-covalently bound to the carrier peptide.

As used herein, a “biologically active molecule” includes any moleculewhich, if transported across the blood-brain barrier, can have abiological effect. Examples of biologically active molecules includepolypeptides, which include functional domains of biologically activemolecules, particular examples include growth factors, enzymes,transcription factors, toxins, antigenic peptides (as for vaccines),antibodies, and antibody fragments. For example, brain derivedneurotrophic factor, fibroblast growth factor (e.g., (FGF)-2 or multipleFGFs), nerve growth factor, neurotrophin (e.g., NT-3 and NT-4/5), glialderived neurotrophic factor, ciliary neurotrophic factor, neurturin,neuregulins, interleukins, transforming growth factor (e.g., TGF-α andTGF-β), vasoactibe intestinal peptide, epidermal growth factor (EGF),erythropoietin, heptocytel growth factor, platelet derived growthfactor, artemin, persephin, netrins, cardiotrophin-1, stem cell factor,midkine, pleiotrophin, bone morphogenic proteins, saposins, semaporins,leukemia inhibitory factor, anti-Aβ, anti-HER2, anti-EGF, anti-nogo A,anti-TRAIL (tumor necrosis factor-related apoptosis-inducing ligand),anti-α-synuclein, anti-htt, anti-prion, anti-West Nile virus,αL-iduronidase, iduronate-2-sulfatase,N-acetyl-galactosamine-6-sulfatase, arylsulfatase B, acid a-glucosidase,and acid sphingomyelinase (See, Pardridge, W. M., Bioconjug. Chem.19(7): 1327-38 2008). Additional examples of biologically activemolecules include oligonucleotides, such as natural or engineeredplasmids, coding DNA sequences, antisense DNA sequences, mRNAs,antisense RNA sequences, RNA is, and siRNAs; carbohydrates; lipids; andglycolipids.

Further examples of biologically active molecules include smallmolecules, including therapeutic agents, in particular those with lowblood-brain barrier permeability. Some examples of these therapeuticagents include cancer drugs, such as daunorubicin and toxic chemicalswhich, because of the lower dosage that can be administered by thismethod, can now be more safely administered. For example, a therapeuticagent can include bevacizumab, irinotecan, zoledronate, andtemozolomide. In another embodiment, the therapeutic agent can include abroad-spectrum antibiotic (e.g., cefotaxime, ceftriaxone, ampicillin andvancomycin); an antiviral agent (e.g., acyclovir); acetazolamide;carbamazepine; clonazepam; clorazepate dipotassium; diazepam; divalproexsodium; ethosuximide; felbamate; fosphenytoin sodium; gabapentin;lamotrigine; levetiracetam; lorazepam; oxcarbazepine; phenobarbital;phenytoin; phenytoin sodium; pregabalin; primidone; tiagabinehydrochloride; topiramate; trimethadione; valproic acid; zonisamide;copaxone; tysabri; novantrone; donezepil HCL; rivastigmine; galantamine;memantine; levodopa; carbidopa; parlodel, permax, requip, mirapex;symmetrel; artane; cogentin; eldepryl; and deprenyl.

Numerous other examples of biologically active molecules will beapparent to the skilled artisan.

Yet another example of a biologically active molecule is an antigenicpeptide. Antigenic peptides can be administered to provide immunologicalprotection when imported by cells involved in the immune response. Otherexamples include immunosuppressive peptides (e.g., peptides that blockautoreactive T cells, which peptides are known in the art).

Polypeptides from a few amino acids to about a thousand amino acids canbe used. In some embodiments, the size range for polypeptides is from afew amino acids to about 250 amino acids (e.g., about 3 to about 250amino acids; about 20 to about 250 amino acids; about 50 to about 250amino acids; about 100 to about 250 amino acids; about 150 to about 250amino acids; about 3 amino acids to about 200 amino acids; about 3 aminoacids to about 150 amino acids; about 3 amino acids to about 175 aminoacids; about 3 amino acids to about 125 amino acids; about 25 aminoacids to about 200 amino acids; about 50 amino acids to about 150 aminoacids; and about 75 amino acids to about 225 amino acids). For anymolecule, size ranges can be up to about a molecular weight of about 1million. In some embodiments, the size ranges up to a molecular weightof about 25,000, and in particular embodiments, the size ranges can beup to a molecular weight of about 3,000.

By “antisense” it is meant a non-enzymatic nucleic acid molecule thatbinds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (proteinnucleic acid; Egholm et al., 1993 Nature 365, 566) interactions andalters the activity of the target RNA (for a review, see Stein andCheng, 1993 Science 261, 1004; Agrawal et al., U.S. Pat. No. 5,591,721;Agrawal, U.S. Pat. No. 5,652,356). Typically, antisense molecules willbe complementary to a target sequence along a single contiguous sequenceof the antisense molecule. However, in certain embodiments, an antisensemolecule may bind to a substrate such that the substrate molecule formsa loop, and/or an antisense molecule may bind such that the antisensemolecule forms a loop. Thus, the antisense molecule may be complementaryto two (or even more) non-contiguous substrate sequences or two (or evenmore) non-contiguous sequence portions of an antisense molecule may becomplementary to a target sequence or both.

RNA interference (RNAi) and short intervening RNA (siRNA) sequences canbe used to modulate (e.g., inhibit) gene expression (see, e.g., Elbashiret al., 2001, Nature, 411, 494 498; and Bass, 2001, Nature, 411, 428429; Bass, 2001, Nature, 411, 428 429; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914). In one embodiment, a siRNA moleculecomprises a double stranded RNA wherein one strand of the RNA iscomplimentary to the RNA of interest. In another embodiment, a siRNAmolecule comprises a double stranded RNA wherein one strand of the RNAcomprises a portion of a sequence of an RNA of interest. In yet anotherembodiment, a siRNA molecule of the invention comprises a doublestranded RNA wherein both strands of RNA are connected by anon-nucleotide linker. Alternately, a siRNA molecule of the inventioncomprises a double stranded RNA wherein both strands of RNA areconnected by a nucleotide linker, such as a loop or stem loop structure.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules (i.e.,molecules that contain an antigen binding site that specifically bindsto a peptide). An antibody can be a monoclonal antibody, a polyclonalantibody, a humanized antibody, a fully human antibody, a single chainantibody, a chimeric antibody, or a fragment thereof. The term “antibodyfragment” of a full length antibody refers to one or more fragments of afull-length antibody that retain the ability to specifically bind to atarget of interest.

An imaging agent, as used herein, can be any chemical or substance whichis used to provide the signal or contrast in imaging. The signalenhancing domain can be an organic molecule, metal ion, salt or chelate,particle (particularly iron particle), or labeled peptide, protein,polymer or liposome.

In some embodiments, the imaging agent is a physiologically compatiblemetal chelate compound consisting of one or more cyclic or acyclicorganic chelating agents complexed to one or more metal ions with atomicnumbers 21-29, 42, 44, or 57-83.

For x-ray imaging, the imaging agent may consist of iodinated organicmolecules or chelates of heavy metal ions of atomic numbers 57 to 83. Insome embodiments, the imaging agent is ¹²⁵I-IgG. Examples of suitablecompounds are described in M. Sovak, ed., “Radiocontrast Agents,”Springer-Verlag, pp. 23-125 (1984) and U.S. Pat. No. 4,647,447.

For ultrasound imaging, the imaging agent can consist of gas-filledbubbles such as Albunex, Echovist, or Levovist, or particles or metalchelates where the metal ions have atomic numbers 21-29, 42, 44 or57-83. Examples of suitable compounds are described in Tyler et al.,Ultrasonic Imaging, 3, pp. 323-29 (1981) and D. P. Swanson, “EnhancementAgents for Ultrasound: Fundamentals,” Pharmaceuticals in MedicalImaging, pp. 682-87. (1990).

For nuclear radiopharmaceutical imaging or radiotherapy, the imagingagent can consist of a radioactive molecule. In some embodiments, thechelates of Tc, Re, Co, Cu, Au, Ag, Pb, Bi, In, and Ga can be used. Insome embodiments, the chelates of Tc-99m can be used. Examples ofsuitable compounds are described in Rayudu GVS, Radiotracers for MedicalApplications, I, pp. 201 and D. P. Swanson et al., ed., Pharmaceuticalsin Medical Imaging, pp. 279-644 (1990).

For ultraviolet/visible/infrared light imaging, the imaging agent canconsist of any organic or inorganic dye or any metal chelate.

For MRI, the imaging agent can consist of a metal-ligand complex of aparamagnetic form of a metal ion with atomic numbers 21-29, 42, 44, or57-83. In some embodiments, the paramagnetic metal is chosen from:Gd(III), Mn(II and III), Cr(III), Cu(II), Dy(III), Tb(III), Ho(III),Er(III) and Eu(III). Many suitable chelating ligands for MRI agents areknown in the art. These can also be used for metal chelates for otherforms of biological imaging. For example, an imaging agent can include:

“Associated with”, as used herein, is meant that the biologically activemolecule or imaging agent is conjugated to the carrier peptide in such amanner that when the carrier peptide crosses the blood-brain barrier,the molecule or agent is also imported. In certain embodiments, thebiologically active molecule or imaging agent is non-covalently bound tothe carrier peptide. For example, the biologically active molecule andthe carrier peptide may be associated through electrostaticinteractions. In other embodiments, the carrier peptide may becovalently bound, either directly or indirectly (e.g., through alinker), to the biologically active molecule or imaging agent.

A linker can be any moiety suitable for linking a carrier peptide to abiologically active molecule. A linker can be bound at the C-terminus,the N-terminus, or both, of a carrier peptide. Additionally, a linkercan be bound to the side chain of a carrier peptide. If a carrierpeptide is bound to multiple linkers, each linker can be different. Alinker can be covalently linked to a side chain of an amino acid, e.g.,lysine, glutamine, cysteine, methionine, glutamate, aspartate,asparagine.

In some embodiments an amino acid side chain can serve as the linker.For example the epsilon amino group (ε-NH₂) can be used to conjugate toa carrier for instance through an amide or thiourea linkage. Similarlythe delta amino group of ornithine (orn), the gamma amino group ofdiaminobutyric acid (dab), or the beta amino group of diamino proprionicacid (dpr) can also act as linkers. These amino acids may be at the C-or N-terminus of the carrier peptide or they may be positioned withinthe carrier peptide sequence.

The complex composed of a biologically active molecule or imaging agentand a carrier peptide can be prepared by any method known by thosehaving ordinary skill in the art. In some embodiments, the carrierpeptide and the biologically active molecule or imaging agent arecombined, incubated at room temperature, and then used. For example,solutions can be prepared of the delivery peptide (K16ApoE (SEQ ID NO:22)) and IgG in PBS (phosphate buffered saline) or OptiMem (commerciallyavailable) or cell culture media without serum in desiredconcentrations. The delivery peptide and IgG can be mixed in the desiredratios and then incubated at room temperature for 30-60 minute.Following incubation, the mixture is ready for injection/delivery.

Combinations of the biologically active molecule and the carrier peptidecan be prepared at a variety of molar ratios. For example, a molar ratioof biologically active molecule to carrier peptide can range from about1:1 to about 1:200 (e.g., 1:2; 1:5; 1:8; 1:10; 1:25; 1:30; 1:40; 1:45;1:50; 1:60; 1:65; 1:70; 1:75; 1:80; 1:90; 1:100; 1:125; 1:135; 1:145;1:150; 1:175; and 1:190). In some embodiments, a molar excess of carrierpeptide to biologically active molecule is used. For example, the molarratio of biologically active molecule to carrier peptide can range fromabout 1:20 to about 1:100 (e.g, 1:50 to about 1:90). In some cases, theratio can be 1:70.

The term “pharmaceutically-acceptable salt” refers to salts whichpossess toxicity profiles within a range that affords utility inpharmaceutical applications. Pharmaceutically unacceptable salts maynonetheless possess properties such as high crystallinity, which mayrender them useful, for example in processes of synthesis, purificationor formulation of compounds described herein. In general the usefulproperties of the compounds described herein do not depend critically onwhether the compound is or is not in a salt form, so unless clearlyindicated otherwise (such as specifying that the compound should be in“free base” or “free acid” form), reference in the specification to acarrier peptide or a complex comprising a carrier peptide should beunderstood as encompassing salt forms of the compound, whether or notthis is explicitly stated.

Suitable pharmaceutically-acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric,salicylic, galactaric and galacturonic acid. Examples ofpharmaceutically unacceptable acid addition salts include, for example,perchlorates and tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts include, forexample, metallic salts including alkali metal, alkaline earth metal andtransition metal salts such as, for example, calcium, magnesium,potassium, sodium and zinc salts. Pharmaceutically acceptable baseaddition salts also include organic salts made from basic amines suchas, for example, N,N-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. Examples of pharmaceutically unacceptable base addition saltsinclude lithium salts and cyanate salts.

All of these salts may be prepared by conventional means from thecorresponding carrier peptide or complex by reacting, for example, theappropriate acid or base with a carrier peptide or complex as describedherein. Preferably the salts are in crystalline form, and preferablyprepared by crystallization of the salt from a suitable solvent. Aperson skilled in the art will know how to prepare and select suitablesalt forms for example, as described in Handbook of PharmaceuticalSalts: Properties, Selection, and Use By P. H. Stahl and C. G. Wermuth(Wiley-VCH 2002).

Methods of Use

Also provided herein are methods of using carrier peptides to transporta biologically active molecule or imaging agent across the blood-brainbarrier of a subject. The method can include administering to thesubject a complex having a biologically active molecule or imaging agentassociated with a carrier peptide, or a pharmaceutically acceptablesalt, as described herein.

A subject can include both mammals and non-mammals. Mammals include, forexample, humans; nonhuman primates, e.g. apes and monkeys; cattle;horses; sheep; rats; mice; pigs; and goats. Non-mammals include, forexample, fish and birds.

Transporting a biologically active molecule can include importing themolecule across the blood-brain barrier.

Further provided herein is a method of treating a brain disorder in asubject. The method can include administering to the subject a complexcomprising a biologically active agent associated with a carrierpeptide, as described herein. In some embodiments, the biologicallyactive molecule is a therapeutic agent. In some embodiments, the braindisorder is chosen from: meningitis, epilepsy, multiple sclerosis,neuromyelitis optica, late-stage neurological trypanosomiasis,Parkinson's, progressive multifocal leukoencephalopathy, De Vivodisease, Alzheimer's disease, HIV Encephalitis, and cancer.

In some embodiments, the brain disorder is a brain cancer, for exampleastrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma),glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma,and congenital tumors; or a cancer of the spinal cord, e.g.,neurofibroma, meningioma, glioma, and sarcoma.

This disclosure also provides a method of imaging the central nervoussystem of a subject. In some embodiments, the method can includeadministering to the subject a complex comprising an imaging agentassociated with a carrier peptide, as described herein, and imaging thecentral nervous system of the subject.

The complex can be administered by any route, e.g., IV, intramuscular,SC, oral, intranasal, inhalation, transdermal, and parenteral.

The complex can be formulated with a pharmaceutically acceptable carrierselected on the basis of the selected route of administration andstandard pharmaceutical practice. The complex may be formulated intodosage forms according to standard practices in the field ofpharmaceutical preparations. See Alphonso Gennaro, ed., Remington'sPharmaceutical Sciences, 18th Edition (1990), Mack Publishing Co.,Easton, Pa. Suitable dosage forms may comprise, for example, tablets,capsules, solutions, parenteral solutions, troches, suppositories, orsuspensions.

For parenteral administration, the complex may be mixed with a suitablecarrier or diluent such as water, an oil (particularly a vegetable oil),ethanol, saline solution, aqueous dextrose (glucose) and related sugarsolutions, glycerol, or a glycol such as propylene glycol orpolyethylene glycol. Solutions for parenteral administration preferablycontain a water soluble salt of the complex. Stabilizing agents,antioxidant agents and preservatives may also be added. Suitableantioxidant agents include sulfite, ascorbic acid, citric acid and itssalts, and sodium EDTA. Suitable preservatives include benzalkoniumchloride, methyl- or propyl-paraben, and chlorbutanol. The compositionfor parenteral administration may take the form of an aqueous ornon-aqueous solution, dispersion, suspension or emulsion.

For oral administration, the complex may be combined with one or moresolid inactive ingredients for the preparation of tablets, capsules,pills, powders, granules or other suitable oral dosage forms. Forexample, the complex may be combined with at least one excipient such asfillers, binders, humectants, disintegrating agents, solution retarders,absorption accelerators, wetting agents absorbents or lubricatingagents.

The specific dose of a complex will, of course, be determined by theparticular circumstances of the individual patient including the size,weight, age and sex of the patient, the nature and stage of the diseasebeing treated, the aggressiveness of the disease disorder, and the routeof administration of the compound.

Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising abiologically active molecule or imaging agent associated with a carrierpeptide. In some embodiments, the biologically active molecule orimaging agent is non-covalently bound to the carrier peptide.

The pharmaceutical compositions provided herein contain a biologicallyactive molecule or imaging agent associated with a carrier peptide in anamount that results in transportation of the biologically activemolecule or imaging agent across the blood-brain barrier, and apharmaceutically acceptable carrier. Pharmaceutical carriers suitablefor administration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration.

The compositions can be, in one embodiment, formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration and intraperitonealinjection, as well as transdermal patch preparation, dry powderinhalers, and ointments (see, e.g., Ansel Introduction to PharmaceuticalDosage Forms, Fourth Edition 1985, 126).

The concentration of the biologically active molecule or imaging agentassociated with a carrier peptide in the pharmaceutical composition willdepend on absorption, inactivation and excretion rates of the compounds,the physicochemical characteristics of the compounds, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art.

The pharmaceutical composition may be administered at once, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by extrapolationfrom in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed compositions.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompounds and derivatives thereof are, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit-dose forms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975.

Dosage forms or compositions containing a biologically active moleculeor imaging agent associated with a carrier peptide in the range of0.005% to 100% with the balance made up with a non-toxic carrier may beprepared. Methods for preparation of these compositions are known tothose skilled in the art. The contemplated compositions may contain0.001%-100% active ingredient, in one embodiment 0.1-95%, in anotherembodiment 75-85%.

Kits

Also provided herein are kits. Typically, a kit includes a carrierpeptide, as described previously. In some embodiments, a kit includes acarrier peptide and a biologically active molecule and/or imaging agent.In certain embodiments, a kit can include one or more delivery systems,e.g., for a biologically active molecule, imaging agent, carrierpeptide, or any combination thereof, and directions for use of the kit(e.g., instructions for administering to a subject). In certainembodiments, a kit can include a biologically active molecule and/or animaging agent, a carrier peptide, and/or a complex of a biologicallyactive molecule or imaging agent and a carrier peptide. In someembodiments, the kit can include a carrier peptide and a label thatindicates that the contents are to be administered to a subject with abiologically active molecule or imaging agent.

EXAMPLES General Materials:

All mice used (B6SJLF1) were female and were purchased from the JacksonLaboratories. Mice were maintained and used following an IRB-approvedprotocol. Bacterial β-galactosidase was purchased from Calbiochem(Catalog #345788). Human IgG and IgM were purchased from Sigma (ProductNumbers I 4506 and 18260 respectively). The 4G8 monclonal antibody (cat#SIG-39220) was purchased from Covance (Emeryville, Calif.). LDL receptorantibody was from abcam (Cat # ab30532).

All peptides were synthesized at the Mayo Proteomic Core Facility.

Preparation of Peptide-Protein Complex for Delivery in the Brain:

Required amount of the peptide and protein were mixed in a final volumeof 300 μL PBS (phosphate buffered saline), and incubated at roomtemperature for 60 minutes. The mixture was vortexed for a few seconds,every fifteen minutes during the incubation period.

The mixture was injected intravenously as a bolus into the lumen of thefemoral vein. This was accomplished using a heat pulled PESO catheter.At the conclusion of the experiment, the mouse was euthanized with alethal dose of sodium pentobarbital. Each mouse was perfused with 10 mLPBS. This perfusion was accomplished through the standard trans cardialmethod. The brain was removed from the skull and positioned to make aninitial coronal slice at −2.0 mm bregma. Subsequently, 25 μm coronalsections were cut on a cryostat and placed on charged slides forstaining of β-galactosidase activity.

Staining for Beta-Galactosidase Enzyme Activity:

Evaluation of β-galactosidase enzymatic activity was accomplished by aninitial 15 minute fixation of the brain sections in 0.25%glutaraldehyde. The slides were washed with 3 changes of PBS for 5minutes each and then rinsed in distilled water for 5 minutes. The brainsections were incubated in X-Gal (0.2%) working solution, pH 7.38, for18 hours at 37° C. in covered containers. Following this incubation thesections were dehydrated and coverslips were applied.

Imaging by microSPECT:

Micro SPECT/CT experiments were conducted on a Gamma Medica X SPECTSystem (GE Healthcare). Human IgG (Sigma) and IgM (Sigma) were labeledto a high specific activity using the Chloramine-T method. 80 μg (500μCi) of each immunoglobulin (corresponds to 0.53 nanomole of IgG and0.13 nanomole of IgM assuming molecular weights of 150 Kd and 600 Kd forIgG and IgM, respectively) was mixed with 70-fold molar excess ofK16ApoE (SEQ ID NO: 22) (as required for experiment) for 1 hour at roomtemperature and was administered in each mouse through the use of acatheter in the femoral vein. Immediately subsequent to the intravenousbolus injection, the mice were imaged every hour for a total of 6 hours.At the completion of the 6 hour time point, each mouse was euthanizedand the systemic blood supply was transcardially perfused with 10 mLphosphate buffered saline, and imaged after 30 minutes.

Example 1—Delivery of Radiolabeled IgG Across the BBB

The delivery of radiolabeled IgG across the BBB in different brainregions in mice with and without the peptide K₁₆ApoE (SEQ ID NO:22) wasevaluated. In addition, the integrity of the BBB after delivery of IgGwith K₁₆ApoE (SEQ ID NO: 22) was examined. 3 μmol of ¹²⁵I-IgG were mixedwith varying amounts of the peptide K₁₆ApoE (SEQ ID NO: 22) (0-55 μmol),and the mixture injected intravenously into the mice. ¹³¹I-IgG notcomplexed with the peptide was injected into the same animals at 59minutes and allowed to circulate for 1 minute subsequent to the initialfemoral vein injection of ¹²⁵I-IgG+K₁₆ApoE (SEQ ID NO: 22). Uptake of¹²⁵I-IgG and ¹³¹I-IgG were measured in different areas of the brainafter 60 minutes.

TABLE 1 +ApoE, +ApoE, +ApoE, +ApoE, IgG, 3 μmol 8 μmol 16 μmol 32 μmol55 μmol PS × 10⁶ (ml/g/s) Cortex 0.13 +− 0.02 0.30 0.47 9.58 15.30Caudate 0.09 +− 0.01 0.35 0.86 16.65 22.60 Hippo 0.17 +− 0.02 0.58 1.0512.52 20.90 Thalamus 0.13 +− 0.02 0.29 1.37 19.60 21.00 Brainstem 0.20+− 0.02 0.45 1.64 22.90 18.40 Cerebellum 0.15 +− 0.02 0.48 0.72 13.4321.60 Vp (ul/g) Cortex 13.53 +− 0.98  21.39 21.22 16.41 25.59 Caudate8.53 +− 0.52 14.44 11.67 12.89 20.10 Hippo 15.13 +− 2.35  17.49 16.9512.67 21.83 Thalamus 13.79 +− 1.09  19.96 21.00 18.34 26.92 Brainstem22.32 +− 1.51  19.02 31.50 20.54 34.60 Cerebellum 20.79 +− 1.24  21.2823.74 19.79 24.31 N (no of mice) 4 2 2 2 1PS values reflect the amount of ¹²⁵I-IgG that has crossed the BBB,whereas Vp values indicate the amount of ¹³¹I-IgG at the BBB but whichhas not crossed the BBB.

As shown in Table 1, the PS values with IgG alone are very small for allthe brain regions, indicating very little transport of the IgG acrossthe BBB. However, the values increased significantly (˜100-fold at 55μmol of K₁₆ApoE (SEQ ID NO: 22)) with increasing amounts of the deliverypeptide.

On the other hand, the Vp values remained virtually unchanged in all thebrain regions examined, indicating ¹³¹I-IgG failed to cross the BBB(since it was not complexed with the peptide), and attesting to thenotion that the BBB was not damaged due to prior exposure to the peptide(during delivery of ¹²⁵I-IgG complexed with K₁₆ApoE (SEQ ID NO: 22)).

Example 2—Delivery of Alexa-IgG Assisted by K₁₆ApoE (SEQ ID NO: 22) intoBrain

In this experiment, AlexaIgG488 was injected into mice with or withoutmixing with K₁₆ApoE peptide (SEQ ID NO:22). Brain sections were made 1hour after delivery. As shown in FIG. 1, the complexed agent showssignificantly brighter contrast and enhancement of brain structurescompared to the uncomplexed AlexaIgG488.

Example 3—Delivery of ApoE Peptide (without K₁₆ Moiety) Across the BBBand Binding of Peptide to LDL Receptors

The experiments will evaluate the ApoE peptide (without the K16 moiety(SEQ ID NO: 4)) crossing of the BBB and whether it will bind/localize toLDL receptors (LDLR) expressed in the brain. An FITC-labeled ApoEpeptide will be synthesized and injected into mice. Brain specimens willbe collected after 1 hour following injection, and slides will beprepared with brain sections of 10 microns thickness. The slides will bestained with antibody against LDLR and evaluated to determine whetherthe signals for LDLR and the ApoE peptide are co-localized.

Example 4—Location of K₁₆ApoE (SEQ ID NO: 22) Following Delivery Acrossthe BBB

The experiments will determine the location of protein delivery by thecarrier peptide (K₁₆ApoE SEQ ID NO: 22)) within the brain. K₁₆ApoE(e.g., SEQ ID NO: 22)+ Alexa-IgG (green fluorescence) conjugate will beprepared by mixing solutions of each at the desired concentrations andincubating the mixture at room temp for 30 minutes. The complex will beinjected into mice and brain specimens collected after 1 hour. Slideswill be prepared as above, and immunostained for GFAP, calbindin andneuregulin. If co-localization of IgG and GFAP is seen, such a resultmay indicate that the delivered IgG reached the astrocytes.Co-localization of IgG and calbindin or neuregulin, however, mayindicate that the delivered IgG reached the neurons.

Example 5—Compromised Integrity of the BBB by K₁₆ApoE (SEQ ID NO: 22)

These experiments will evaluate whether the K₁₆ApoE (e.g., SEQ IDNO:22), alone or following formation of a complex, compromises theintegrity of the BBB. A complex with K₁₆ApoE (SEQ ID NO: 22)+AlexaRedIgGwill be prepared and injected into mice. After 1 hour uncomplexedAlexaGreenlgG will be injected. Brain specimens will be collected 1 hourafter the second injection, and slides will be prepared using 10 micronsections. The sections will be visualized using confocal microscopy. Inother mice, the reverse the experimental approach will be implementedusing AlexaGreenlgG complexed with K₁₆ApoE (SEQ ID NO: 22) anduncomplexed AlexaRedIgG. If the peptide does not compromise the BBB,then only red fluorescence will be seen in cells in the brain. However,if the peptide does compromise the BBB, then both red and greenfluorescence will be seen, and vice versa.

Example 6—Evaluation of microSPECT

A microSPECT machine can image and quantify radioactivity in varioustissues in live animals under anesthesia. If K₁₆ApoE (SEQ ID NO: 22)indeed carries a protein (radioactive) across the BBB, then there shouldbe more radioactivity in the brain of a mice injected with such apeptide-protein complex than in the brain of a mice injected with theradioactive protein alone.

IgG 4.1 (an antibody against the amyloid precursor protein (APP)) wasradioiodinated. IgG4.1 alone or previously complexed with K₁₆ApoE (SEQID NO:22) was injected in a number of mice. The mice were anesthesizedand the brain was imaged with a microSPECT machine at 1 hour intervals.Perfusion was done for some mice after six hours of quantitative imagingfollowed by imaging after 30 minutes.

The radioactivity measured at each time point for each mice was plottedagainst time (FIG. 2). Seven animals were used in total, they arenumbered M3-M9. M3 and M4 mice were not perfused. Two mice (M5 and M7)receiving IgG 4.1 were perfused after 6 hours of imaging, and three mice(M6, M8 and M9) receiving the IgG complexed with the delivery peptidewere perfused after 6 hours of imaging followed by imaging at 6.5 hours.

It is clear from the data presented in FIG. 2 that ˜2-fold moreradioactivity was registered in the brains of mice delivered with theIgG complexed with the peptide than the IgG alone. This differenceincreased to nearly 5-fold when the animals were perfused.

Radioactivity in the brains of mice receiving IgG alone indicated IgGmolecules were retained in the capillaries. Increased accumulation ofradioactivity in the brains of mice receiving the IgG complexed with thedelivery peptide indicated IgGs were present in the capillaries as wellas having crossed the BBB and retained in the brain. If the IgG crossesthe BBB and is retained in brain cells, then perfusion of the brainsshould eliminate IgGs in the capillaries but IgGs that have crossed theBBB should still be retained, which is essentially seen for mice M6, M8and M9, providing strong indication that the peptide helps carry theprotein (IgG) through the BBB into the brain.

Example 7—Delivery of a Protein Across the BBB

To evaluate the potential of K16ApoE (SEQ ID NO: 22) for delivering aprotein across the BBB, the enzyme β-galactosidase was injected in micewith or without prior mixing with K16ApoE (SEQ ID NO: 22) (at a proteinto peptide molar ratio of 1:70). The experiment was repeated more than adozen times. In this series of experiments, intense β-galactosidaseactivity was observed in mice brain when the enzyme-peptide mix wasinjected and brain slices were prepared for enzyme activity staining 6hours after injection, whereas no activity was seen when the enzyme wasinjected alone (FIG. 3A). High-magnification scans of the images ofβ-galactosidase-stained sections in the brain showed that the enzyme wasdelivered in virtually every area of the brain, and stained bothastrocytes and neurons, however, pyramidal cells in the hippocampusshowed much weaker staining for β-galactosidase activity compared tocells in other areas of the brain. The weak β-galactosidase activitystaining in the pyramidal cells does not appear to be due to lowexpression of LDLR in mouse hippocampus since cells in this regiondisplay strong immunohistochemical staining for the receptor. It isspeculated that either X-gal or 5-bromo-4-chloroindole or both somehowleach out of this region faster than other regions of the brainresulting to a faint signal for β-galactosidase activity.

To assess if the delivery of β-galactosidase in the brain via K16ApoE(SEQ ID NO: 22) is LDLR-mediated, β-galactosidase staining in liver,brain, kidney, heart and lung after injection of the enzyme with andwithout mixing with K16ApoE (SEQ ID NO: 22) was also evaluated. The mostintense staining was observed in liver and brain, followed by stainingin lung, heart and kidney (FIG. 3B-G). This pattern of uptake ofbeta-galactosidase by K16ApoE (SEQ ID NO: 22) in various organsapproximately follows the pattern of reported LDLR expression patternsuggesting that delivery of β-galactosidase through K16ApoE (SEQ ID NO:22) is LDLR-mediated.

Example 8—Delivery of IgG and IgM Using K16ApoE

Since many potentially therapeutic proteins against brain-associateddisorders are immunoglobulins, delivery of IgG and IgM through K16ApoE(SEQ ID NO: 22) in mouse brain was evaluated by micro-single photonemission computed tomography (microSPECT) imaging. For this,radioiodinated human IgG (Sigma, product # I 4506) and human IgM (Sigma,product # I 8260) were mixed with K16ApoE (SEQ ID NO: 22) and injectedintravenously. Anesthetized animals were then subjected to imaging for 6hours at 1 hour interval. Cardiac perfusion was then performed, afterwhich final imaging was done at 30 minutes post-perfusion. Results fromthese experiments provide a number of indications (FIG. 4): First, itappears that peak delivery of both IgG and IgM into brain happens around2 hours post-injection of the peptide-protein mix, after which uptake ofthe proteins in the brain remains relatively unchanged up to 6 hours(maximum time point tested). Second, cardiac perfusion appears to removemost of the radioactive protein in the vasculature, concomitantlyincreasing the difference between apparent brain-uptake of the proteinwith and without the carrier peptide (˜5-fold). Finally, the resultssuggest that about 1% of the injected immunoglobulins are transportedinto the brain by K16ApoE (SEQ ID NO: 22) under the experimentalcondition. It is interesting to note that the extent of delivery of theradiolabeled IgG and IgM into kidney, liver/spleen and heart remainedvirtually unchanged irrespective of whether the proteins were deliveredwith or without the carrier peptide. Cardiac perfusion also did notchange the accumulation of radioactivity in these organs, implying thattransport of proteins in these organs does not depend upon crossing of abiological barrier.

Example 9—Efficacy of Delivered Therapeutic

For its activity, β-galactosidase does not need to bind with anymolecular entity in the brain. However, for all therapeutic purposes,the delivered molecule in the brain must be able to recognize and bindwith its cognate target molecule. To test this premise, an antibodyagainst amyloid beta peptide (4G8 from Cavance) was delivered viaK16ApoE (SEQ ID NO: 22) in the brains of mice which model forAlzheimer's disease. This antibody is known to recognize amyloid betaplaques. The results indicate that the antibody delivered in this mannerlabeled the amyloid plaques in the brain of these mice as well asidentifyed the plaques with the antibody through standardimmunohistochemistry (FIG. 5). Similar results were also obtained withanother antibody (4.1 IgG, 32) known to label these plaques.

Example 10—Transportation of Protein without Damage to BBB

A transporter that efficiently carries a protein in the brain should notdo so by impairing the integrity of the BBB. To evaluate if K16ApoE (SEQID NO: 22) impairs the integrity of the BBB, the peptide was firstinjected intravenously, then injected β-galactosidase at different timeintervals. The control mice received the enzyme mixed with the peptide.Mice were perfused and sacrificed 6 hours after β-galactosidaseinjection, brain slices were made and prepared for β-galactosidasestaining. Results presented in FIG. 6 show increasingly weakerβ-galactosidase activity in the brains of mice receiving β-galactosidaseat 1, 5 and 10 minutes after injection of K16ApoE (SEQ ID NO: 22) and novisible staining thereafter. These results indicate that most of theinjected K16ApoE (SEQ ID NO: 22) becomes bound with proteins and cellsin the circulation, and this binding becomes virtually complete within10 minutes after which no free K16APoE (SEQ ID NO: 22) remains incirculation. Free K16ApoE (SEQ ID NO: 22) that remains at early timepoints becomes bound with beta-galactosidase and carries the enzyme inthe brain. The peptide by itself and/or being bound with bloodproteins/cells did not seem to affect the BBB as no β-galactosidaseenzyme activity was seen in the brain from 10 min to 4 hours.

Example 11—Necessity of Covalent Linking of Hydrophillic Amino AcidChain and Carrier Peptide

The necessity of covalent linking of the K16 (SEQ ID NO: 4) and ApoE(SEQ ID NO: 13) peptide moieties was evaluated for non-covalenttransport of a protein across the BBB. For this, β-galactosidase wasmixed with either the ApoE peptide, the peptide K16 (SEQ ID NO: 4), ApoEplus K16 (SEQ ID NO: 4) or with K16ApoE (SEQ ID NO: 22), and injected inmice. Brain sections from these mice show β-galactosidase staining onlyin slides from mice receiving β-galactosidase mixed with K16ApoE (SEQ IDNO: 22), indicating that linking of the K16 (SEQ ID NO: 4) and the ApoEpeptide moieties is critical for transport of a protein in anon-covalent manner (FIG. 7).

Example 12—Testing Mutant ApoE Peptides for their Potential asNon-Covalent Transporters of Proteins Across the BBB

β-galactosidase was mixed with equal amounts of various peptidetransporters and injected into mice. Slides were prepared using 25 μmsections of the mice brains one hour after injection followed bydevelopment of enzyme staining (FIG. 8). The following peptides wereevaluated:

A: (SEQ ID NO: 126) KKKK KKKK KKKK KKKK LRLR LANH LRKL RKRL LRDA; B:(SEQ ID NO: 127) KKKK KKKK KKKK KKKK LRLR LASH LRKL RKRL LRDL; C:(SEQ ID NO: 128) KKKK KKKK KKKK KKKK LRVR LANH LRKL RKRL LRDL.As shown in FIG. 8, mutant A was worse than the wild-type ApoEblood-brain barrier agent, while mutants B and C were able to transportthe β-galactosidase at least as well as wild-type or better.

Example 13—Time Course for β-Galactosidase Delivery in Mouse Brain withK16ApoE (SEQ ID NO: 22)

In each mouse, 1.38 nanomoles of β-galactosidase was mixed with 89nanomoles of K16ApoE (SEQ ID NO: 22) and injected intravenously. 25 μmbrain slices were prepared for staining for β-galactosidase activity atindicated time points (FIG. 9). FIG. 8 provides the following timepoints:

A—Beta-galactosidase, no peptide, 6 h.B—Beta-galactosidase+ peptide, 1 h.C—Beta-galactosidase+ peptide, 2 h.D—Beta-galactosidase+ peptide, 5 h.E—Beta-galactosidase+ peptide, 10 h.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. (canceled)
 2. A method of transporting a nerve growth factor acrossthe blood-brain barrier of a subject, the method comprisingadministering to the subject a complex, or a pharmaceutically acceptablesalt thereof, comprising the biologically active molecule or imagingagent associated with a carrier peptide; wherein the carrier peptidecomprises the sequence:X_(n)—[B]_(m) or a pharmaceutically acceptable salt thereof, wherein: Xis a hydrophilic amino acid; B is a blood-brain barrier agent; n is aninteger from 4 to 50; and m is integer from 1 to
 3. 3. The method ofclaim 2, wherein each X is independently chosen from arginine,asparagine, aspartic acid, glutamic acid, glutamine, lysine, serine,threonine, and tyrosine.
 4. The method of claim 3, wherein n is chosenfrom 4, 8, 12, 16, and
 20. 5. The method of claim 4, wherein n is
 16. 6.The method of claim 2, wherein m is
 1. 7. The method of claim 2, whereinthe nerve growth factor is non-covalently bound to the carrier peptide.8. The method of claim 2, wherein the blood-brain barrier agentcomprises a receptor binding domain of an apolipoprotein is chosen fromthe receptor binding domain of ApoA, ApoB, ApoC, ApoD, ApoE, ApoE2,ApoE3, and ApoE4.
 9. The method of claim 2, wherein B comprises thesequence: (SEQ ID NO: 29)L-R-X1-R-X2-X3-X4-H-L-R-X5-X6-X7-K-R-L-X8-R-D-X9

wherein: X1 is selected from the group consisting of A, L, S, and V; X2is selected from the group consisting of L and M; X3 is selected fromthe group consisting of A and S; X4 is selected from the groupconsisting of N, S, and T; X5 is selected from the group consisting of Kand N; X6 is selected from the group consisting of L, M, and V; X7 isselected from the group consisting of R and P; X8 is selected from thegroup consisting of L and M; and X9 is selected from the groupconsisting of A and L.
 10. The method of claim 9, wherein the carrierpeptide comprises the sequence: (SEQ ID NO: 143)[X]_(n)-L-R-X1-R-X2-X3-X4-H-L-R-X5-X6-X7-K-R-L-X8-R- D-X9

wherein: X is lysine.
 11. The method of claim 10, wherein the carrierpeptide comprises the sequence: (SEQ ID NO: 19)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 20)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 21) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 22)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 23)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 46)K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 47)K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 48) K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 49)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 50)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A ; (SEQ ID NO: 51)K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 52)K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 53) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 54)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 55)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 56)K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 57)K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 58) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 59)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 60)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-T-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 61)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 62)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L- R-D-A;(SEQ ID NO: 63) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 64)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 65)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 66)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 67)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M- R-D-A;(SEQ ID NO: 68) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 69)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 70)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 71)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 72)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 73) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 74)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 75)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 76)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 77)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L- R-D-A;(SEQ ID NO: 78) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 79)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 80)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 81)K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 82)K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 83) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 84)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 85)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-M-S-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 86)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 87)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L- R-D-A;(SEQ ID NO: 88) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 89)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 90)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-V-R-K-R-L-L-R-D-A; (SEQ ID NO: 91)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 92)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L- R-D-A;(SEQ ID NO: 93) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 94)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 95)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-M-R-K-R-L-L-R-D-A; (SEQ ID NO: 96)K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 97)K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 98) K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 99)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 100)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-A-R-M-A-S-H-L-R-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 101)K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 102)K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M- R-D-A;(SEQ ID NO: 103) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 104)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 105)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 106)K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 107)K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M- R-D-A;(SEQ ID NO: 108) K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 109)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 110)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-S-R-L-A-S-H-L-R-K-L-R-K-R-L-M-R-D-A; (SEQ ID NO: 111)K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 112)K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L- R-D-A;(SEQ ID NO: 113) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 114)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 115)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-S-S-H-L-P-K-L-R-K-R-L-L-R-D-A; (SEQ ID NO: 116)K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 117)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M- R-D-A;(SEQ ID NO: 118) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 119)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 120)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-K-M-R-K-R-L-M-R-D-A; (SEQ ID NO: 121)K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 122)K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L- R-D-A;(SEQ ID NO: 123) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 124)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 125)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-S-H-L-R-N-L-P-K-R-L-L-R-D-A; (SEQ ID NO: 131)K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 132)K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L- R-D-L;(SEQ ID NO: 133) K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 134)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 135)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-L-R-L-A-S-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 136)K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 137)K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L- R-D-L;(SEQ ID NO: 138) K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; (SEQ ID NO: 139)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L; or (SEQ ID NO: 140)K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-L-R-V-R-L-A-N-H-L-R-K-L-R-K-R-L-L-R-D-L.


12. A method of treating a brain disorder in a subject, the methodcomprising administering to the subject a complex, or a pharmaceuticallyacceptable salt thereof, comprising a nerve growth factor associatedwith a carrier peptide, wherein the carrier peptide comprises thesequence:X_(n)—[B]_(m) wherein: X is a hydrophilic amino acid; B is a blood-brainbarrier agent; n is an integer from 4 to 50; and m is integer from 1 to3.
 13. The method of claim 12, wherein the brain disorder is chosenfrom: meningitis, epilepsy, multiple sclerosis, neuromyelitis optica,late-stage neurological trypanosomiasis, Parkinson's, progressivemultifocal leukoencephalopathy, De Vivo disease, Alzheimer's disease,HIV Encephalitis, and cancer.